Although high-throughput technologies have enhanced our understanding of bacterial virulence gene regulation, the development of antibiotics that target the regulatory systems that are essential for bacterial pathogenesis has not been extensively pursued. We hypothesize that small molecules inhibiting the conserved PhoP virulence regulon may constitute effective antibiotics. The PhoP regulon is an essential regulator of the genes required for intracellular survival and virulence of a number of pathogens and has been best characterized in the model organism of Salmonella enterica serovar Typhimurium. Here, we propose to develop a high throughput molecular screening (HTS) assay to identify chemical inhibitors of the PhoP regulon. In particular, our strategy will be designed to screen for small molecules that simultaneously inhibit the expression of reporter genes from PhoP-activated promoters, while increasing the expression of genes from PhoP-repressed promoters. In Specific Aim 1, we will develop a series of recombinant PhoP-activated and PhoP-repressed promoter-reporter fusions, and quantify the expression of these reporters in serovar Typhimurium grown in PhoP-inducing and non-inducing conditions. In Specific Aim 2, we will configure the assay for HTS by selecting a single PhoP-activated and a single PhoP-repressed promoter with the highest signal-to- background ratios and piloting the assay in 96 and 384 well formats. In this proposal we also outline a detailed sequential strategy for the secondary evaluation and prioritization of active compounds identified in the initial HTS screen. Multi-drug resistant bacteria are important causes of global morbidity and mortality and have the capacity to overcome our current biodefense, all of which necessitate the development of novel antibiotic classes. The proposed assay may identify small molecules with unique antibiotic properties and a novel mechanism of action against intracellular pathogens including the drug- resistant cause of typhoid fever, a major cause of morbidity and mortality worldwide, as well the causative agent of the plague, Yersinia pestis, a significant bioterrorist threat. In future studies, compounds identified using the proposed assays may be developed into a new class of drugs targeted for the treatment of many bacterial infections, including several category A and B priority pathogens. [unreadable] [unreadable]